Authors' Affiliations: Divisions of Oral Maxillofacial Pathology & Radiology and Oral Maxillofacial Surgery & Anesthesiology, College of Dentistry, The Ohio State University; The Ohio State University Comprehensive Cancer, Columbus, Ohio; Departments of Oral & Maxillofacial Pathology and Oral & Maxillofacial Surgery, School of Dentistry, University of Louisville, Kentucky; Departments of Diagnostic Sciences and Oral and Maxillofacial Surgery, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, North Carolina; and Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, WisconsinAuthors' Affiliations: Divisions of Oral Maxillofacial Pathology & Radiology and Oral Maxillofacial Surgery & Anesthesiology, College of Dentistry, The Ohio State University; The Ohio State University Comprehensive Cancer, Columbus, Ohio; Departments of Oral & Maxillofacial Pathology and Oral & Maxillofacial Surgery, School of Dentistry, University of Louisville, Kentucky; Departments of Diagnostic Sciences and Oral and Maxillofacial Surgery, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, North Carolina; and Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin

Authors' Affiliations: Divisions of Oral Maxillofacial Pathology & Radiology and Oral Maxillofacial Surgery & Anesthesiology, College of Dentistry, The Ohio State University; The Ohio State University Comprehensive Cancer, Columbus, Ohio; Departments of Oral & Maxillofacial Pathology and Oral & Maxillofacial Surgery, School of Dentistry, University of Louisville, Kentucky; Departments of Diagnostic Sciences and Oral and Maxillofacial Surgery, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, North Carolina; and Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin

Authors' Affiliations: Divisions of Oral Maxillofacial Pathology & Radiology and Oral Maxillofacial Surgery & Anesthesiology, College of Dentistry, The Ohio State University; The Ohio State University Comprehensive Cancer, Columbus, Ohio; Departments of Oral & Maxillofacial Pathology and Oral & Maxillofacial Surgery, School of Dentistry, University of Louisville, Kentucky; Departments of Diagnostic Sciences and Oral and Maxillofacial Surgery, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, North Carolina; and Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin

Authors' Affiliations: Divisions of Oral Maxillofacial Pathology & Radiology and Oral Maxillofacial Surgery & Anesthesiology, College of Dentistry, The Ohio State University; The Ohio State University Comprehensive Cancer, Columbus, Ohio; Departments of Oral & Maxillofacial Pathology and Oral & Maxillofacial Surgery, School of Dentistry, University of Louisville, Kentucky; Departments of Diagnostic Sciences and Oral and Maxillofacial Surgery, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, North Carolina; and Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin

Authors' Affiliations: Divisions of Oral Maxillofacial Pathology & Radiology and Oral Maxillofacial Surgery & Anesthesiology, College of Dentistry, The Ohio State University; The Ohio State University Comprehensive Cancer, Columbus, Ohio; Departments of Oral & Maxillofacial Pathology and Oral & Maxillofacial Surgery, School of Dentistry, University of Louisville, Kentucky; Departments of Diagnostic Sciences and Oral and Maxillofacial Surgery, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, North Carolina; and Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin

Authors' Affiliations: Divisions of Oral Maxillofacial Pathology & Radiology and Oral Maxillofacial Surgery & Anesthesiology, College of Dentistry, The Ohio State University; The Ohio State University Comprehensive Cancer, Columbus, Ohio; Departments of Oral & Maxillofacial Pathology and Oral & Maxillofacial Surgery, School of Dentistry, University of Louisville, Kentucky; Departments of Diagnostic Sciences and Oral and Maxillofacial Surgery, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, North Carolina; and Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin

Authors' Affiliations: Divisions of Oral Maxillofacial Pathology & Radiology and Oral Maxillofacial Surgery & Anesthesiology, College of Dentistry, The Ohio State University; The Ohio State University Comprehensive Cancer, Columbus, Ohio; Departments of Oral & Maxillofacial Pathology and Oral & Maxillofacial Surgery, School of Dentistry, University of Louisville, Kentucky; Departments of Diagnostic Sciences and Oral and Maxillofacial Surgery, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, North Carolina; and Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin

Authors' Affiliations: Divisions of Oral Maxillofacial Pathology & Radiology and Oral Maxillofacial Surgery & Anesthesiology, College of Dentistry, The Ohio State University; The Ohio State University Comprehensive Cancer, Columbus, Ohio; Departments of Oral & Maxillofacial Pathology and Oral & Maxillofacial Surgery, School of Dentistry, University of Louisville, Kentucky; Departments of Diagnostic Sciences and Oral and Maxillofacial Surgery, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, North Carolina; and Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin

Authors' Affiliations: Divisions of Oral Maxillofacial Pathology & Radiology and Oral Maxillofacial Surgery & Anesthesiology, College of Dentistry, The Ohio State University; The Ohio State University Comprehensive Cancer, Columbus, Ohio; Departments of Oral & Maxillofacial Pathology and Oral & Maxillofacial Surgery, School of Dentistry, University of Louisville, Kentucky; Departments of Diagnostic Sciences and Oral and Maxillofacial Surgery, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, North Carolina; and Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin

Authors' Affiliations: Divisions of Oral Maxillofacial Pathology & Radiology and Oral Maxillofacial Surgery & Anesthesiology, College of Dentistry, The Ohio State University; The Ohio State University Comprehensive Cancer, Columbus, Ohio; Departments of Oral & Maxillofacial Pathology and Oral & Maxillofacial Surgery, School of Dentistry, University of Louisville, Kentucky; Departments of Diagnostic Sciences and Oral and Maxillofacial Surgery, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, North Carolina; and Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin

Authors' Affiliations: Divisions of Oral Maxillofacial Pathology & Radiology and Oral Maxillofacial Surgery & Anesthesiology, College of Dentistry, The Ohio State University; The Ohio State University Comprehensive Cancer, Columbus, Ohio; Departments of Oral & Maxillofacial Pathology and Oral & Maxillofacial Surgery, School of Dentistry, University of Louisville, Kentucky; Departments of Diagnostic Sciences and Oral and Maxillofacial Surgery, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, North Carolina; and Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin

Authors' Affiliations: Divisions of Oral Maxillofacial Pathology & Radiology and Oral Maxillofacial Surgery & Anesthesiology, College of Dentistry, The Ohio State University; The Ohio State University Comprehensive Cancer, Columbus, Ohio; Departments of Oral & Maxillofacial Pathology and Oral & Maxillofacial Surgery, School of Dentistry, University of Louisville, Kentucky; Departments of Diagnostic Sciences and Oral and Maxillofacial Surgery, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, North Carolina; and Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin

Authors' Affiliations: Divisions of Oral Maxillofacial Pathology & Radiology and Oral Maxillofacial Surgery & Anesthesiology, College of Dentistry, The Ohio State University; The Ohio State University Comprehensive Cancer, Columbus, Ohio; Departments of Oral & Maxillofacial Pathology and Oral & Maxillofacial Surgery, School of Dentistry, University of Louisville, Kentucky; Departments of Diagnostic Sciences and Oral and Maxillofacial Surgery, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, North Carolina; and Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin

Abstract

Purpose: Approximately 30% higher grade premalignant oral intraepithelial neoplasia (OIN) lesions will progress to oral cancer. Although surgery is the OIN treatment mainstay, many OIN lesions recur, which is highly problematic for both surgeons and patients. This clinical trial assessed the chemopreventive efficacy of a natural product-based bioadhesive gel on OIN lesions.

Translational Relevance

Oral squamous cell carcinoma (OSCC), which arises from its premalignant precursor oral intraepithelial neoplasia (OIN), is a worldwide health problem. Although not all OIN lesions will transform, approximately 30% of the higher grade lesions progress to OSCC. Furthermore, OIN lesions often recur despite complete surgical excision. Numerous chemoprevention studies have therefore attempted to induce regression in or prevent progression of OIN lesions. Modest success and dose-limiting toxicities were often the outcomes of these clinical trials. The majority of previous OIN clinical studies relied upon systemic chemopreventive agent administration. In contrast, this study assessed the effects of a food-based (freeze-dried black raspberries) bioadhesive gel on OIN lesions. Our results, which demonstrate BRB gel significantly reduces LOH, lesional size, and histologic grade in OIN lesions without any toxicities combined the absence of such effects in the placebo gel cohort, are favorable.

Introduction

Oral squamous cell carcinoma (OSCC) is a worldwide health problem and one of the most challenging-to-treat human malignancies (1). This is due to the insidious nature of its early disease, reliance upon surgery as the primary treatment modality, and the difficulty of achieving locoregional disease control (1, 2). Many patients with OSCC die from massive local recurrence or second primary tumors (3, 4). Also, despite treatment innovations like inductive chemotherapy and radiation intensification programs, OSCC survival rates remain among the lowest for solid tumors (4, 5). Those patients fortunate enough to be cured often encounter major esthetic and functional issues with their face and mouth (6).

Previous OSCC prevention trials have used a variety of chemopreventive compounds including retinoic acid and its derivatives (10), green tea and associated polyphenols (11), and the cyclooxygenase (COX)-2 inhibitor, celecoxib (12), as well as combination of celecoxib and the EGF receptor (EGFR) inhibitor erlotinib (13). Unfortunately, the majority of these previous studies, which relied on systemic agent administration, resulted in modest to negligible chemopreventive effects (10–12) and induced appreciable toxicities (13).

Black raspberries (BRB) were selected as the trial chemopreventive due to their success in our previous in vitro and in vivo chemoprevention studies (14, 15) and because of their chemopreventive rich composition, which includes four anthocyanins (the predominant BRB polyphenols which provide appreciable chemopreventive impact; ref. 16), ellagic acid, ferulic acid, coumaric acid, and quercetin, phytosterols in addition to folic acid and selenium (17, 18). Furthermore, local BRB anthocyanin metabolism has the potential to sustain local chemopreventive effects. For example, deglycosylation generates highly active anthocyanidins that are converted to the more stable metabolite protocatechuic acid which is capable of undergoing local enteric recycling (19).

Our pilot clinical trial results showed topical application of a bioadhesive gel that contained 10% w/w freeze-dried BRBs (0.5 g, q.i.d. for 6 weeks, n = 20) significantly reduced LOH in OIN lesions, modulated epithelial gene expression toward terminal epithelial differentiation, and significantly reduced OIN levels of COX-2 (14, 15). These pilot trial data also revealed appreciable interpatient variations in BRB gel responsiveness (14, 15). A subsequent study was conducted to characterize intraoral bioactivation and retention of BRB anthocyanins in healthy human volunteers to help elucidate mechanisms attributable for these variations in responsiveness (19). As BRB metabolizing enzymes are primarily distributed in the surface epithelia, OIN lesional keratinocytes are well positioned to benefit from BRB bioactivation (19). Also, there is extensive interdonor heterogeneity with regard to epithelial levels of enzymes responsible for BRB bioactivation and local enteric recycling (19). It is therefore logical to speculate that patients with higher levels of BRB bioactivation and local enteric recycling enzymes would derive greater chemopreventive benefit due to sustained levels of BRB chemopreventives at the OIN lesional site. A component of this current study was designed to further investigate this BRB metabolism-therapeutic efficacy question.

The current study expanded upon our pilot study as it was multi-University based (Ohio State, North Carolina at Chapel Hill and Louisville, KY), incorporated both a BRB containing and a BRB-devoid (placebo) gel and doubled treatment time (3 months vs. 6 weeks). Therapeutic efficacies of BRB-containing and placebo gels (0.5 g applied q.i.d. for 3 months) were determined by their effects on: (i) clinical lesional size, (ii) OIN histopathologic grade, and (iii) LOH status at putative tumor suppressor gene loci associated with OIN progression to OSCC [3p14 (FHIT), 9p21 (INK4a/ARF), 9p22 (IFN-α), and 17p13 (p53)]. Pretreatment OIN biopsies were used to microscopically confirm a premalignant diagnosis and provide baseline biomarkers. Additional protein profiling assays were conducted to assess the contribution of differentiation and metabolic-local recycling enzymes on BRB gel efficacy. Results from the current study confirm that therapeutic efficacy is attributable to the BRB constituents as only the BRB gel-treated lesions showed significant therapeutic responses as indicated by improvement in lesional size and histologic grade and reduction of LOH events. In contrast, placebo gel-treated lesions significantly increased in size and did not show significant changes in histologic regression or LOH status over the study duration.

Materials and Methods

Berry gel manufacturing

Freshly harvested BRB from Strums Berry Farm were immediately freeze dried and ground into powder at Oregon Freeze Dry Inc. BRB powder was shipped on dry ice to JR Chemical, LLC for gel preparation using Current Good Manufacturing Practices. The BRB gel composition used in this trial was identical to our pilot study (14). The placebo formulation replaced 10% BRB with 10% w/w sucrose and food colorants (FD&C Red #40 and FD&C Blue #1) to provide the matching dark blue-black color. With the exception of sucrose and food colorants, the placebo gel was identical to the 10% FBR gel. The 10% FBR-containing and placebo gels are manufactured by JR Chemical LLC.

Covance Laboratories Inc. analyzed the BRB powder. Berry constituents were found to closely replicate (≤15%) the component distributions in the pilot gel batch (14). Gel stability and bioburden tests for both the BRB and placebo gels were conducted by Bioscreen Testing Services, Inc. at 0, 1, 3, 6, 9, 12, 18, and 24 months, which corresponded to the time frame for gel usage.

Clinical trial inclusion/exclusion criteria

Investigational New Drug approval was obtained from the U.S. Food and Drug Administration (IND#109774). Institutional Review Board (IRB) approvals were obtained from the IRBs at all three participating Universities, that is, Ohio State, University of North Carolina, Chapel Hill and Louisville (Trial registration ID: NCT01192204). Forty adults (see Table 1) were consented for trial participation. Inclusion criteria were microscopically confirmed premalignant oral epithelial lesions, no use of tobacco products for 6 weeks prior and during the 3-month study, and no previous history of cancer (except for basal cell carcinoma of the skin). Participants were screened before entrance into and during (10–12 days recall intervals) the trial for no tobacco use compliance via unannounced saliva testing for nicotine (NicAlert, JANT Pharmacal Corporation). Trial exclusion criteria were previous or current history of nonbasal cell cancer, use of tobacco products, and either a microscopic diagnosis of no premalignant change or OSCC in the pretrial biopsy. The National Cancer Institute (Bethesda, MD) common toxicity criteria version 4.0 was used. All participants returned to clinic every 10 to 14 days for continued assessment. Used gel tubes were returned and new gels were dispensed.

Assessment of lesional clinical size

Clinical photographs of the participants' OIN lesions [pretreatment-time 0, week 1 (biopsy follow-up, baseline for subsequent measurements), mid-study (6 weeks), and immediately before final biopsy] were taken with a calibrated measuring device (Puritan) placed parallel to the long axis of the lesion. Acquired clinical images were analyzed using ImagePro 6.2 software (Media Cybernetics). Lesional sizes were normalized to square millimeters (mm2) according to the following formula: lesional size mm2 = pixels of lesional area × 100/(pixels of 1 centimeter unit on the calibration device in the same image)2. The remaining lesional area after the initial biopsy and before gel treatment was the pretreatment size. Posttreatment lesional size was the residual lesional area after 3 months gel treatment and just before the final biopsy.

PCR amplification and detection of allelic imbalance

Genomic DNA isolated from the LCM samples was amplified using the predesigned primers with a 5′ fluorescent label on the forward primer (Life Technologies). The microsatellite markers selected for LOH analyses and their corresponding loci and associated genes were: 3p14 [D3S1007 (VHL), D3S1234 (FHIT)], 9p21 [D9S171, D9S1748 (P16/CDKN2A), D9S1751 (P16)], 9p22 (IFN-α), and 17p13 [D17S786 (P53) and TP53]. Twenty microliter PCR mixture, which contained 5 μL of genomic DNA, 2 μL of primer mix (0.5 μmol/L of each primer), 10 μL of AmpliTaq Gold 360 master mix (Life Technologies), and 3 μL of nuclease-free water, was amplified using a iCycler thermal cycler (Bio-Rad). PCR conditions were: 95°C for 5 minutes followed by 40 cycles of 95°C for 15 seconds, 59°C for 30 seconds, 72°C for 45 seconds, and a final elongation step of 72°C for 7 minutes. Fragment analyses were performed at the OSU Plant Microbe Genomics Facility using the Applied Biosystems 3730 sequence analyzer. One microliter of PCR product DNA was added to 9 μL HiDi (formamide; Applied Biosystems, Inc.) and 0.2 or 0.4 μL (volume dependent upon DNA concentration) GeneScan-500 LIZ Size Standard (Applied Biosystems, Inc.) for analysis. Both automatic and manual (enabled editing) settings for allele identification (GeneMapper v4.0, Applied Biosystems) were used to analyze electropherogram data. Peak intensities ≤50 RFUs (relative fluorescent units) were excluded for being within background. Microsatellite marker peak patterns and allele sizes were established from normal DNA (15). Connective tissue control samples with only one allele were deemed “not interpretable” (NI). In several instances, the PCR amplification products in the normal connective tissue for a particular patient/marker combination were inadequate to allow LOH determination and were designated as “not available” (NA). LOH determinations were made using a modification of the protocol established by Canzian and colleagues (21) as described by Shumway and colleagues (15), using an increased level of stringency (>50% reduction in peak intensity) to accept the presence of LOH (21). Baseline OIN LOH status was determined from the initial biopsy and then compared with LOH status in treated lesional tissue.

Evaluation of differentiation and local enteric recycling enzymes and cornified envelope precursor proteins by immunoblot analyses

Statistical analyses

Two-tailed Mann–Whitney U tests were used to compare pretreatment baseline parameters, that is, size, histologic grade and LOH events in the BRB and placebo gel cohorts. A Wilcoxon matched-pairs signed rank test was used to compare the pre and posttreatment histologic grades, clinical lesional sizes, and LOH events. Cumulative treatment responses (effects on lesional size, histologic grade, and LOH events) were evaluated by two-tailed unpaired t test. A Fisher exact test was used to compare distribution of responsiveness between the BRB and placebo gel treatments. Associations between two therapeutic evaluative parameters were determined by a Spearman rank correlation. Relationships among size, histologic grade, and LOH used multiple regression analysis. Normality of data determined the use of parametric versus nonparametric analyses.

Results

Patient demographics and comparable pretreatment baseline parameters

Forty patients participated in this study. Participants' ages ranged from 44 to 77 years (mean 62.2 ± 1.8) and 32 to 78 years (mean 57.7 ± 2.9) in the BRB and placebo gel cohorts, respectively. A majority of the patients never smoked (55% in BRB and 67% placebo gels) and tongue was the most common OIN site in both groups. The gender distribution in the BRB gel cohort was 78.2% (15 women) and 31.8% (7 men), whereas the placebo group was evenly distributed (50% each, 9 women, 9 men; Table 1). Thirty of the participants had OIN lesions (16 in BRB-72.7% and 14 in the placebo-77.7%) that were recalcitrant to surgery, and had recurred multiple times (2–8) at the same site before trial participation. Twelve BRB (54.5%) and three placebo gel (16.7%) participants had a history of multiple premalignant oral epithelial lesions dispersed throughout their mouth, consistent with a diagnosis of proliferative verrucous leukoplakia (Table 1). No histopathologic evidence of concurrent human papillomavirus infection, for example, koilocytic change in lesional epithelial cells was observed in any of the pre or posttreatment lesional tissue biopsies.

Effect of gel treatment on lesional size. Histograms A (BRB gel) and B (placebo gel) depict pre- and posttreatment lesional size (mm2) of individual subjects' OIN lesions. The lesional sizes of subject A4 in the BRB group and subject P11 in the placebo group were not included as the extensive distribution and confluent nature of their dysplastic lesions made accurate measurement impossible. C, the mean pretreatment lesional sizes of both groups were statistically comparable. Intragroup pre/post lesional sizes significantly decreased in the BRB gel treatment group, whereas significantly increased in the placebo group. D, intergroup comparison reviewed a significant difference between the BRB and placebo groups. E and F, this clinical photograph of subject A6 was taken before the initial biopsy of the crisply defined rhomboid white plaque close to commissure. The area of the residual lesion remained after initial biopsy was measured as the pretreatment biopsy size. F, clinical photograph at the same site after 3 months BRB gel treatment and immediately before the final biopsy. The remaining lesion had completely regressed and the buccal mucosa at the treatment site had a normal clinical appearance.

Effect of gel treatment on LOH. A and B, pre- and posttreatment detected LOH events of individual subjects in BRB gel and placebo gel groups, respectively. C, intragroup and intergroup statistical analyses. Mean LOH events significantly decreased in the BRB gel treatment group, whereas changes in the placebo group were statistically insignificant. The baseline LOH events of both pretreatment groups were statistically comparable. D, classification of subjects as per treatment effects on LOH events. E and F, representative genotyping data depict an LOH event occurred on marker D9S171 in subject A19′s pretreatment samples [loss of one allele (al 154) in the epithelium tissue (E), compared with the two alleles (al 154 and al 162) in the patient's matched normal connective tissue (F)]. G and H, the lost allele (al 154) of D9S171 was recovered in the epithelial tissue (G) of the same patient after 3 months of BRB gel treatment, and the ratio of peak heights in epithelium (G) was comparable with the connective tissue sample (H).

No deleterious effects were observed in either the BRB gel or placebo gel cohorts and compliance was excellent

Although topical application of a gel could result in deleterious effects, for example, contact mucositis or superimposed Candidiasis, no participant experienced any treatment-associated complications. Furthermore, as determined by the minimal residual gel in the returned gel tubes (>95% dose used), patient compliance was high.

BRB gel significantly decreased OIN lesional clinical size

Following 3 months of BRB gel application, 16 of the 21 BRB-treated lesions decreased in size (P = 0.0019) for an average overall size decrease of 26%. In contrast, 17 of the 19 placebo gel-treated lesions increased in clinical lesional size (P = 0.0395) with an average increase of 18% (see Table 1; Fig. 1A–C). Although none of the placebo gel patients experienced complete lesional regression, two BRB gel patients had 100% lesional resolution. These individuals' mucosa returned to a clinically (and histologically) normal and healthy appearance (Fig. 1D).

Densitometry analyses of protein immunoprecipitation studies. Five proteins associated with keratinocyte terminal differentiation (TGase 1, high-molecular weight involucrin, low-molecular weight involucrin, loricrin, cytokeratin 10/13) and two with BRB metabolism (UDP-Glc dehydrogenase and UGT1A) were evaluated in pre- and posttreatment biopsy samples of those lesions that had adequate tissue. All densitometry results were normalized to the percentage of sample-matched pancytokeratin level, which reflected the proportion of epithelium tissue in each sample. Previous studies have confirmed the epithelial distribution of UDP-Glc dehydrogenase and UGT1 (19). Responder and nonresponder were defined according to the cumulative responsiveness score (responder ≥1, nonresponder ≤0). A and B, pre- and posttreatment protein levels in the BRB group. Responders demonstrated a trend of possessing higher level of associated proteins relative to the nonresponders. C and D, pre- and posttreatment protein levels in the placebo group. No statistical significance was identified.

Longer term follow-up reveals OIN lesional recurrence in both cohorts

Thirty eight of the 40 trial patients were available for the requisite 3-month posttrial evaluation; one patient each was lost to follow up in the BRB and placebo gel cohorts. Three months after trial cessation, 6 of 22 BRB and 7 of 17 placebo patients had visible evidence of lesional recurrence at the former treatment sites (Table 1). Only one patient (P6) lesion had a clinically significant lesional recurrence that merited biopsy scheduling at the 3-month recall.

Patients were then returned to their previous oral health care providers for subsequent care. As many of these patients are treated by oral maxillofacial surgeons who use the pathology biopsy services at the trial institutions, longer term follow-up (4 to 31 months postfinal study biopsy) was available for many trial participants (see Supplementary Table S2). Longer term poststudy biopsies were received from 8 of the 22 BRB gel and 6 of the 18 placebo gel cohort patients. Two BRB gel cohort patients' lesions (25%) regressed to nonpremalignant states, four biopsies remained stable (50%), and two lesions progressed (Supplementary Table S2). One of these progressive patients underwent progression of two histologic grades, whereas another lesion returned to its pretrial histopathologic grade. Three of the six placebo gel patients' lesions remained stable, three lesions progressed. Two of the three progressive lesions from the placebo gel cohort increased one histologic grade, whereas the third individual's lesion (P11) progressed from atypia (pretreatment) to moderate dysplasia (postplacebo gel application) to OSCC in the 12 months posttrial (Supplementary Table S2).

Discussion

Topical application of a 10% BRB gel resulted in significant reduction in size, histologic grade, and LOH events in OIN lesions. The absence of comparable results following placebo gel application strongly supports BRB's chemopreventive impact and dispels other contributions such as topical stimulation during gel application or hydrogel constituents.

Following BRB gel application, 41% of participants achieved a decrease in lesional grade, 4.5% a lesional grade increase, and 54.5% retained stable OIN histology. Although the 41% grade decrease is identical to our pilot BRB gel trial (14), the percentage of OIN lesions that histologically progressed are 5-fold lower in the current study; findings that likely reflect the doubled treatment time of the current trial (14). A range of histologic responsiveness has been observed in other OIN clinical trials (10–13). The “most responsive” group (13 cis-retinoic acid) in the combination 13 cis-retinoic acid, β-carotene, and retinyl palmitate trial demonstrated a 30% histologic reduction (10). In the green tea extract study, 33% (3 of 9) obtained histologic regression in the most responsive but toxicity-associated highest dosing group (1,000 mg/m2 t.i.d.), with an overall, a 21.4% rate of histologic regression (6 of 28; ref. 11). The celecoxib trial did not include histopathologic assessment (12). Recently, the effects of combined administration of celecoxib (400 mg b.i.d) with escalating doses (50, 75, 100 mg q.d.) of the EGFR inhibitor erlotinib were assessed on premalignant oral and laryngeal lesions (13). Comparison of baseline histology to final biopsies (obtained at 3, 6, or 12 months) in the 7 evaluable patients showed 43% complete regression (3/7, one laryngeal, two oral lesions), 14% (1/7) partial regression, 29% (2/7) stable disease, and 14% progression (1/7; ref. 13). No placebo group was included (13). Complementary biomarker studies demonstrated significant reduction of EGFR and p-ERK in biopsies that showed histologic improvement (13). Erlotinib dose escalation was accompanied by toxicities including oral mucositis, rash, anemia, sepsis, and elevated liver enzymes; effects that the authors acknowledged as unfavorable for primary chemoprevention (13). Because oral dysplastic lesions have been shown to be less treatment responsive than oropharyngeal lesions (33), separate reporting of oral and laryngeal lesions seems warranted in more definitive studies (33).

Because of the effects of first pass metabolism and the need for the agent to perfuse from the connective tissue papilla to the avascular epithelia, bioavailability is often challenging for systemically administered OIN chemopreventives (34). Attempts to address the bioavailability challenge by dose escalation are often accompanied by toxicity (11, 13). Furthermore, it is interesting that neither this recent pharmacokinetic study (13) nor any of the previously cited OIN trials determined the levels of parent compound(s) and/or metabolites achieved at the treatment site (10–12). Before conducting our pilot clinical trial, we established that topical BRB gel application provides a pharmacologic advantage at human oral mucosa (35).

LOH-mediated inactivation of one of the two alleles of tumor suppressor genes followed by silencing of the second allele via promoter methylation or point mutation is a putative and probable tumorigenic mechanism (36, 37). Clinical data, which demonstrate a higher risk of malignant transformation in OIN lesions that harbor LOH at tumor suppressor gene loci, support this premise (15, 38). Pretreatment LOH events detected in the current study were lower than those detected in either our pilot trial and in two investigations conducted by Mao and colleagues (39, 40). These variations likely reflect differences in baseline lesion histology, LOH analytical methods, and microsatellite markers evaluated (15, 39, 40). Consistent with previous investigations (15, 37, 39, 40), allelic imbalances in this current study were highest at the 9 p loci. BRB gel treatment significantly reduced allelic imbalances, whereas placebo gel did not significantly affect LOH status. To our knowledge, our pilot BRB pilot gel trial (15) and this current study are the only OIN chemoprevention trials to demonstrate a significant reduction in LOH occurrence. We speculate that these data reflect removal of LOH harboring keratinocytes from the proliferative pool via BRB-mediated induction of apoptosis and/or differentiation, as previously demonstrated by in vitro studies (31).

Although preliminary, our protein profiling data suggest that BRB gel responsiveness may be associated with lesional keratinocytes' differentiation and local enteric recycling enzymatic capacities. Provided additional studies substantiate these findings, baseline protein levels could be used in a “personalized medicine approach” to identify OIN lesions with a high probability of responsiveness. Notably, the highest pretreatment levels of differentiation-associated proteins were detected in the most responsive BRB gel cohort.

The observed posttreatment lesional recurrences were not surprising, particularly because more than 70% of the patients enrolled in this trial had histories of multiple recurrences of the OIN lesion selected for treatment. These recurrences, which are consistent with retention of genetically altered, long-lived stem cells at the lesional site, emphasize the need for effective, nontoxic, long-term chemoprevention strategies.

This study shares shortcomings with other OIN trials. First is the dynamic nature of OIN lesions. OIN lesions with a homogenous clinical appearance can still demonstrate molecular and/or histologic heterogeneity (41). Also, as a result of ongoing epithelial turnover baseline to posttreatment biopsies are not direct comparisons. Instead, these measurements assess the effects of treatment on transient amplifying and more mature lesional keratinocytes over time. Furthermore, low patient numbers precluded our ability to determine whether or not clinical site or baseline histologic grade affected therapeutic responsiveness. Another common clinical trial challenge is the interpatient variation in responsiveness. Our data imply that these results reflect differences in local tissue absorption and the extensive variability in human oral mucosal metabolic bioactivation, local enteric recycling, and keratinocyte differentiation-associated enzymes (19).

A recent editorial by a well-respected oral cancer chemoprevention researcher helps to place our results in perspective (42). The presence of LOH at specific chromosomal loci (3p and 9p) was acknowledged as the most consistent molecular marker of oral cancer risk (38, 41). Also discussed was the inability to identify a standard systemic treatment protocol despite numerous, costly OIN chemoprevention clinical trials (42). In this context, BRB gel outcomes, which include significant reduction in OIN LOH events, lesional size, and histologic grade without adverse effects, are favorable.

Disclosure of Potential Conflicts of Interest

S.R. Mallery has ownership interest (including patents) in BEB gel patent. No potential conflicts of interest were disclosed by the other authors.

Grant Support

This work was supported by NIH NCI RC2 CA148099.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Acknowledgments

The authors thank the expertise provided by Ohio State University College of Dentistry's Oral Pathology histotechnologists, Mary Lloyd and Mary Marin, and the excellent patient support provided by Wendy Lamm, CDA, Dental Research Clinic Coordinator, School of Dentistry, University of North Carolina.

Footnotes

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).